Stability Evaluation and Pharmacokinetic Profiling of Vepdegestrant in Rodents Using Liquid Chromatography-Tandem Mass Spectrometry

Abstract

Vepdegestrant (formerly ARV-471), a novel proteolysis-targeting chimera (PROTAC), targets estrogen receptor alpha (ERα) for degradation, offering a promising option to treat advanced ER-positive breast cancer. We developed and validated a sensitive and rapid liquid chromatography-tandem mass spectrometry method to quantify vepdegestrant in rodent plasma using bavdegalutamide (formerly ARV-110) as an internal standard. Plasma samples were prepared with protein precipitation using acetonitrile and analyzed using reverse-phase C18 columns and a mobile phase of 10 mM ammonium formate in distilled water and acetonitrile. The method demonstrated linearity from 1 to 1000 ng/mL in mouse and rat plasma, meeting all validation criteria, and successfully applied to in vivo and in vitro studies. Pharmacokinetic analysis revealed low-to-moderate clearance (313.3, 1053 mL/h/kg) and oral bioavailability (17.91, 24.12%) of vepdegestrant in mice and rats, respectively. It was unstable in buffer solutions across pH 2-10 and in phosphate-buffered saline (pH 7.4), likely due to adsorption, but remained stable in mouse and rat plasma at varying temperatures. In liver microsomes, vepdegestrant exhibited moderate stability in rats but was stable in mice, dogs, and humans. These findings enhance the understanding of pharmacokinetic properties of vepdegestrant supporting further development of PROTAC drugs.

Keywords: ARV-471; LC-MS/MS; PROTAC; pharmacokinetics; stability; validation; vepdegestrant.

Figure 1

Product ion mass spectra for (a) vepdegestrant and (b) bavdegalutamide (internal standard; IS).

Figure 2

High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) chromatograms of vepdegestrant and the bavdegalutamide (IS) for mouse plasma. (a) Blank mouse plasma absent vepdegestrant and the IS; (b) Zero sample, mouse plasma containing only IS (300 ng/mL); (c) Mouse plasma spiked with 1 ng/mL (lower limit of quantification; LLOQ) vepdegestrant and IS; (d) Incurred sample 15 min following intravenous injection of 2 mg/kg vepdegestrant to mice.

Figure 3

HPLC-MS/MS chromatograms of vepdegestrant and the bavdegalutamide (IS) for rat plasma. (a) Blank rat plasma absent vepdegestrant and the IS; (b) Zero sample, rat plasma containing only IS (300 ng/mL); (c) Rat plasma spiked with 1 ng/mL (LLOQ) vepdegestrant and IS; (d) Incurred sample 15 min following intravenous injection of 2 mg/kg vepdegestrant to rats.

Figure 4

The remaining amount of vepdegestrant (%) in buffer solution (pH 2, ●; pH 4, □; pH 6, ▲; pH 7.4, ○; pH 8, ■; pH 10, △). Data represent the mean ± standard deviation (n = 3).

Figure 5

The remaining amount of vepdegestrant (%) in mouse plasma at 4 °C (●), mouse plasma at 25 °C (□), mouse plasma at 37 °C (▲), rat plasma at 4 °C (○), rat plasma at 25 °C (■), and rat plasma at 37 °C (△). Data represent the mean ± standard deviation (n = 3).

Figure 6

The remaining amount of vepdegestrant (%) in mouse (●), rat (□), dog (▲), and human (○) liver microsomes (a) and the remaining amount of vepdegestrant (%) in rat liver microsomes with NADPH (●), without NADPH (□), and in PBS (▲) (b). Data represent the mean ± standard deviation (n = 3).

Figure 7

Plasma concentration–time profiles of vepdegestrant following intravenous administration at 2 mg/kg (●) and oral administration at 5 mg/kg (□) to male ICR mice (a) and SD rats (b). Data represent the mean ± standard deviation (n = 6 for mice and 5 for rats).